Figure 5.

(A) Survival of hind-limb or skin allografts in mice transplant model. B6 mice received hind-limb allografts from B10.A donors and were treated with a short-term co-stimulation blockade plus rapamycin protocol. Full-thickness skin allograft recipients with the same immunosuppressive treatment and hind-limb allograft recipients without treatment were served as controls. (B) Donor cell percentage in PBL of B10.A to B6 hind-limb allograft recipients. The percentages of H2D^d+ cells in PBL of recipients were analyzed by flow cytometry. (C, D) The deletion of donor-specific TCR Vβ clones and the absence of donor-specific antibodies in tolerant recipients. (C) TCR Vβ expression profiles in PBL of the tolerant recipients were analyzed at POD 120 by flow cytometry and expressed as the percentages of TCR Vβ5.1/5.2⁺, Vβ8⁺, Vβ11⁺ cells in CD4⁺ cells The Naïve B6 and naïve B10.A mice were used as controls. (D) Donor-specific IgM, IgG antibodies in PBL of recipients were measured at POD 120. Splenocytes from the donor B10.A mouse were first incubated with sera collected from tolerant recipients, or recipients that underwent graft rejection, or naïve B6 mice, and then stained with fluorochrome-conjugated anti-mouse IgM, or IgG, and CD4 antibodies. Splenocytes were analyzed by flow cytometry for anti-donor antibodies expression on CD4⁺ T cells. Results were displayed as mean fluorescence intensity (MFI) ± SEM. (E) Survival of skin allografts in Rag1^-/- mice receiving CD4⁺CD25^- T cells. CD4⁺CD25^- T cells (5x10⁶) harvested from the splenocytes of tolerant B6 mice were i.v. injected to the B6.Rag1^-/- mice. Naïve B6 CD4⁺CD25^- T cells were used as controls in adoptive transfer. Recipient Rag1^-/- mice were further challenged with skin allografts from B10.A (donor), BALB/c (third party), and naïve B6 mice (native control). Skin grafts from naïve B6 mice were accepted by all adoptive transferred B6.Rag1^-/- mice recipients. (F) Survival of skin allografts in nude mice receiving thymus transplant. Thymus lobes from the tolerant B10.A-B6 chimeric mice were transplanted into the subrenal capsule of the B6 nude mice. At POD14, skin allografts from B10.A (donor), BALB/c (third party), and naïve B6 mice were transplanted simultaneously to the same thymus-bearing B6 nude mice.